Molecular Basis for Nitric Oxide Dynamics and Affinity with Alcaligenes xylosoxidans Cytochrome c´

Abstract

The bacterial heme protein cytochrome c from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman timeresolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 ؎ 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.Nitric oxide (NO) acts as a second messenger in several physiological systems (1, 2), is involved in the production of several cytotoxic chemical species and nitrosative stress (3), and appears as an intermediate in the denitrification process (4). Cells have, thus, developed numerous heme proteins whose diverse functions involve nitric oxide binding and/or release. In some bacteria heme sensors were recently discovered (5) with a femtomolar affinity for NO, whereas cytochromes cЈ able to bind NO are found in many nitrogen-fixing, denitrifying, and photosynthetic bacteria (6). Although the physiological role the bacterial cytochrome cЈ from Alcaligenes xylosoxidans (AXCP) 2 is not yet established, the homologous cytochrome cЈ from Rhodobacter capsulatus has been shown to increase the resistance of this bacteria against NO toxicity (7,8). Thus, the bacterial cytochromes cЈ are inferred to disclose a particular behavior regarding NO dynamics and affinity. How heme proteins interact with NO and control its reactivity is ultimately related to their structure and the heme surroundings. A wide range of affinity and kinetic constants can be observed that correlates with the molecular dynamics of NO within the protein core. A striking example of this diversity is offered when comparing the NO dynamics governed by the enzyme NO synthase (9), the endogenous cellular source, and the NO receptorsoluble guanylate cyclase (10) (sGC) whose heme cofactor has a somewhat opposite role. Nitrosylated heme proteins usually form 6-coordinate complexes having histidine and NO as axial ligands at the proximal and distal sides of the heme, respectively (6c-NO-His), but some become 5-coordinate (5c) with NO (5c-NO) like sGC (11, 12) and AXCP (13,14). These latter proteins have an overall structure that presumably controls the strain upon the proximal histidine. Although sGC and AXCP possess different sequences and tertiary structures, they share the pro...

Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase

“…It must be emphasized that because the NO geminate recombination to the 4c heme is ultrafast (τ gem = 7.5 ps), numerous cycles of photodissociation/recombination can occur for the same molecules during the 6-ns pulse used. This phenomenon, already observed for cytochrome c′ (28,33), results in a higher apparent dissociation yield so that kinetics in subsequent decades appear with a higher amplitude relative to the initial peak than in measurements using a femtosecond excitation pulse (Fig. 2C).…”

“…The dissociation of NO yields a 4c heme, as observed by the bleaching at 397 nm (Fig. 2B) to which NO geminately rebinds without energy barrier from within the heme pocket with τ gem = 7.5 ps (25,26), a property observed in other 4c heme proteins (28,29). Accordingly, the induced absorption centered at 428 nm due to the 4c heme decreases rapidly, and a new maximum appears, centered at 433 nm, meaning that a second species was formed and decays more slowly.…”

“…Because it has been shown that the position of the heme Fe atom does not have an impact on the rate of NO geminate rebinding, either to the 4c heme (28) or to the 5c-His heme (31), we attribute this high reactivity to its electronic configuration. Indeed, our transient Raman spectroscopy studies have suggested that the 4c transient heme is a high-spin species with single-electron occupancy of both d(z 2 ) and d(x 2 − y 2 ) orbitals (28). Whereas NO geminate rebinding experiences no energy barrier, the very small energy barrier for His 105 rebinding is most probably due to the rotation of the side chain, which accounts for the 10-fold larger time constant τ His = 70 ps.…”

Quaternary Structure Controls Ligand Dynamics in Soluble Guanylate Cyclase

“…If the structural conformation of the heme domain is not strictly the same for full-length protein versus isolated domain, then the diatomic ligand dynamics will be modified. It is established that the dynamics of a diatomic ligand within the protein core is highly sensitive to the heme pocket conformation and to strains on the heme iron through the proximal histidine, which imposes energy barriers to diatomic motion (15)(16)(17). Here, we compared the dynamic interaction of NO and CO within the full-length sGC and the isolated human heme domain of the ␤ 1 -subunit, restricted to the first 190 amino acids, referred to as ␤ 1 (190) hereafter.…”

Modulation of ligand–heme reactivity by binding pocket residues demonstrated in cytochrome c' over the femtosecond–second temporal range

“…This is demonstrated by the high proportion of geminate recombination after excitation (~ 99%) on an ultrafast time scale (τ = 7 ps) 18. Laser‐flash photolysis studies have shown that only ~ 1% of the population releases NO to the surrounding bulk solvent, with rebinding of the proximal histidine to the heme Fe 20.…”